Assistant Professor Worcester Polytechnic Institute Worcester, Massachusetts, United States
Introduction: Exclusive breastfeeding for 6 months is the recommended feeding method for an infant with health benefits for both infant and breastfeeding parent. Despite approximately 90% of birthing parents in the U.S. initiating breastfeeding after birth, currently only 25% of infants are exclusively breastfed at 6 months. Challenges with breastfeeding that frequently lead to early weaning include concerns of milk production, but only an estimated 5% of birthing parents have a physiological barrier to producing sufficient milk. Clinically, a diagnosis of insufficient milk production is a diagnosis of exclusion and cannot be made until 1-2 weeks postpartum. Milk production occurs within the cell in response to the polypeptide hormone prolactin and regulated by the signal transducer and activator of transcription 5 (STAT5) protein. To develop an assay that can distinguish between perceived and real insufficient milk production requires a cell culture model that can vary lactation output.
Materials and
Methods: We performed a 2D culture of MCF7 mammary cells at 3 different temperatures (37°C, 38°C, and 39°C) with (lactation) and without (control) lactogenic culture media on 35 mm well plates with a glass slide cover on the bottom and seeded at 2 x 104 cells per cm2. The control media was prepared according to ATCC protocol with the addition of 1% (v/v) GlutaMAX supplement and 1% (v/v) Penicillin Streptomycin. The lactation media consisted of the control media with dexamethasone (1 ug/mL) and prolactin (3 ug/mL). Media was changed every 2 days until Day 8 when the glass slides with attached cells were removed, stained for the milk protein β-casein and nuclei, and imaged. Lactation production was quantified with a custom MATLAB code to count the number of pixels with β-casein and nuclei.
Results, Conclusions, and Discussions: All cells cultured in the lactation media showed signs of lactation (Figure 1). The concentration of β-casein in the 37°C and 38°C cultures was significantly greater compared with the 39°C cultures. The ratio of β-casein-stained pixels to nuclei-stained pixels was 106.05%, 6.71%, and 1.281% for 37°C, 38°C, and 39°C cultures, respectively, indicating that more β-casein was released and moved outside the nuclei of the cells towards the cell wall. Prolactin caused hypertrophy of cells, so cell size was measured and evaluated with a one factor ANOVA test. While the lactating cells were statistically larger (p < 0.001) at 65.65 um2 than the control cells at 46.56 um2, no significance was found between the sizes of lactating cells at different cultures. This indicates that prolactin binds to the cells in less-than-ideal conditions. Clinically during pregnancy, an increase in breast size due to lactogenic hormones and cell hypertrophy is usually predictive of lactation success. However, this study shows increased cell size is not a good biomarker of lactation success. Similarly, all the lactating cells had long connections between cells compared to the cobblestone morphology of the control group. In conclusion, we successfully designed a cell culture protocol that can vary lactation output for human mammary cells by varying the temperature of the culture. This protocol can be modified for other mammary cell lines in both humans and other mammals to study insufficient milk production at a cellular level and develop an assay for clinical use.
Acknowledgements (Optional): The author would like to acknowledge the assistance of Leithsa Dimanche, Jazmyn Ewing, Caroline Major, and Taina Quiñones for their assistance with this project.
